This years major accomplishments are in the following areas: 1) Reciprocal negative regulation between Lmx1a and Lmo4 is required for inner ear formation The structurally complex mammalian inner ear requires a multitude of signaling molecules to mediate its formation. Identifying some of these key molecules and their functions during inner ear formation will someday lead to the design of better strategies to alleviate balance and hearing disorders resulted from structural defects of the inner ear. During embryogenesis, secreted molecules that activate developmental processes are often regulated by specific endogenous antagonists. Likewise, activities of transcription factors are also regulated by other transcription regulators. For example, the LIM-family of transcription factors (LIM-TF) are competed by LIM-only proteins (LMO) for binding to co-factors within the same transcription complex. However, LMO cannot bind DNA itself and thus function as negative regulators of LIM-TF functions. In the past year, we published a paper showing that the reciprocal negative regulation between a LIM-TF, Lmx1a, and one of the LMO-only proteins, Lmo4, function to shape the formation of various components of the inner ear. 2) Directional selectivity of afferent neurons in zebrafish neuromasts is regulated by Emx2 in presynaptic hair cells All sensory end-organs need to be properly connected to the central nervous system by sensory neurons before sensory inputs can be interpreted in a meaningful manner. Understanding the mechanisms involved in this proper wiring during development is important from a functional standpoint. The lateral line system of aquatic animals, which functions to detect water pressure, is comprised of neuromasts that are studded along the surface of the animals body. Each neuromast consists of two groups of sensory hair cells, in which their hair bundles are oriented in opposite direction to each other, along either the anterior-posterior or dorsal-ventral axis of the body. It has been shown in zebrafish neuromasts that hair cells with opposite orientations are segregated by the neurons that innervate them. While a single neuron can innervate multiple neuromasts, it only innervates hair cells that are oriented in the same direction. The mechanisms whereby afferent neurons select hair cells of the same orientation was not known. Previously, we have shown that the transcription factor Emx2 is expressed in only half of the hair cells within a zebrafish neuromast. The presence of Emx2 in these hair cells causes them to reverse their hair bundle orientation from the default location by approximately 180 degrees and thus establishes the bidirectional hair bundle orientation pattern in a neuromast. Based on these results, we asked whether the directional selectivity of the afferent neurons is also dependent on Emx2. Using gain- and loss-of Emx2 function zebrafish mutants, we demonstrated that in the Emx2 knockout mutants, in which all hair cells within a neuromast are oriented in one rather than two directions, the neuronal innervation pattern was altered. Likewise, similar disruption of innervation pattern was observed in Emx2 gain-of-function mutants. These results indicate that Emx2 expressed in sensory hair cells regulates selection of post-synaptic neurons. Furthermore, although both hair bundle orientation and selection of neuronal targets require Emx2, these two cellular processes are independently regulated by transcription targets of Emx2. This study is now published in the journal eLife. 3) Vestibular evoked potential is generated by the striola, a specialized central zone of the maculae The vestibular system of the inner ear plays an important role in maintaining our sense of balance. Understanding how head movements and positional information are being coded by the vestibular sensory organs and relayed to the brain is important from a clinical and therapeutic perspective. The two vestibular sensory organs, maculae of the utricle and saccule, are responsible for detecting linear acceleration. A specialized region known as the striola is present in the center of each macula. A defining feature of the striola is the calyceal nerve endings of afferent neurons that often encase multiple hair cells. The precise function of these complex calyces is not known but based on physiological properties of the hair cells and sensory neurons in the striola, this region is thought to be responsible of rapid signaling. In the past several years, we have generated a viable mouse mutant, Cyp26b1 condition knockout (cKO), in which the striola is missing. Cyp26b1, a Retinoic acid (RA) degradation enzyme, is expressed in the prospective striolar region during development and it functions to reduce the amount of RA emanating from the sensory tissues surrounding the striola. Based on the molecular, cellular and physiological analyses, the striola in Cyp26b1 cKO mice exhibit extrastriolar-like properties. As a result, these cKO mutants show remnants or absence of vestibular evoked potential (VsEP), which is a standard test for assessing macular function of the vestibule. Our results indicate that the specialized striola, which only constitutes approximately 15% of the total area of a macula, is primarily responsible for eliciting the VsEP in response to jerk stimuli. We are currently investigating the behavioral deficits of these mutants. 4) Role of Sonic Hedgehog in mediating spiral ganglion formation in the mouse inner ear Despite the sophisticated mammalian cochlea tailoring to the detection of sound from the environment, this sound information cannot be received by the brain without the proper wiring of the spiral ganglion (SG) neurons that connect the two. Therefore, understanding how neurons in the SG establish their connections during development will pave the way for treating cochlear neuropathy, which is one of the causes underlying neurosensory hearing loss. Previously, we have shown that Sonic hedgehog (Shh) secreted by the SG is important for mediating growth of the cochlear duct and timing of cochlear hair cell differentiation. The absence of Shh in the SG causes a shortened cochlear duct and premature differentiation of cochlear hair cells from an apical to basal direction along the cochlear duct, opposite to the direction of hair cell differentiation observed in a normal cochlea. Additionally, the mutant SG is also much reduced in size. However, Shh is only expressed in a subpopulation of the SG throughout its development. To address the nature of the Shh-positive neurons and their role in SG formation, we traced the lineage of Shh expressing cells in the developing SG. We found that although most, if not all, SG neurons are part of the Shh lineage, only a subpopulation of the developing SG expresses Shh at a given time. We further determined that this subpopulation of Shh-positive cells changes over time and only represents nascent neurons that just exited from the cell cycle. These nascent neurons downregulate Shh expression as they differentiate. We also found that Shh in the nascent SG neurons signal to the adjacent neuroblasts. Given the reduction in the size of mutant SG, we postulate that Shh regulates the proliferation and cell cycle exit of adjacent neuroblasts via its receptor Patched. We are currently investigating the molecular identity of Shh-positive neurons in the developing SG using single-cell RNA seq approach. Using this approach, we hope to identify the role of Shh in regulating the neuronal development of SG neurons.